The purification of insulins or insulin derivatives on lipophilically modified (reversed phase) silica gels is known from analytic separation procedures and has been used successfully for many years in high pressure liquid chromatography (HPLC) (W S Welinder et al., J. Chrom., 361, (1986) 357-367). On an analytical scale protein quantities in the .mu.g region are added to a column of steel, glass or plastic that is filled with modified silica gel, and subsequently eluted with a mixed fluid stream (mainly acidic, aqueous buffer solutions with a constant or variable concentration of organic solvents). In a separation of this type the protein loading is substantially less than 30 .mu.g/ml of column volume.
The insulins from previous chemical conversions, for example from strongly acidic ester cleavages or enzymatic (trans)peptidation processes and from crystallization purifications, usually contain concomitant substances with similar properties. They can be purified by ion exchange chromatography by the choice of particular pH values, if adequate electric charge differences exist (U.S. Pat. No. 4,129,560). The disadvantage of this method lies in the dilution effect, and the consequent loss of valuable material in the supernatants during the working up of the precipitates, in the relatively long cycle time and in the fact that the total recovery and thus the yield is less.
Insulin preparations with depot properties frequently contain protamines which extend the length of action of insulins. Protamines are arginine-rich proteins which are obtained from roe. Quality tests on depot insulin preparations regularly showed an inexplicable loss of the depot properties after the preparation had been stored for some time. Such insulin preparations are completely unsuitable for the patient, since their use can readily lead to insulin overdosing, which may sometimes lead to the death of the patient following hypoglycemic shock.
Precise investigations of these insulin preparations showed that the loss of the depot properties is caused by a slow degradation of the protamines by traces of proteases. Additionally, it was found that the major quantity of proteases in these preparations arises from the enzymatic transpeptidation of porcine insulin to human insulin ester/ether or from proteolytic processes for the cleavage of insulin-like precursors. Although a large part of the proteases, for example trypsin, is separated off by the commonly-employed chromatographic procedures, it is evident that residual quantities of proteases do remain in the insulin preparations and lead there to the loss of depot properties.
In the preparation of insulin by genetic engineering the serine situated at position A9 of the insulin is also acetylated by the microorganism as a by-product, so that an insulin derivative is formed that is acetylated at the serine hydroxyl group. This insulin derivative has thus far only been observed in the preparation of insulin with Escherichia coli using genetic engineering. The complete separation of this acetylated insulin derivative from human insulin has so far only been achieved at the cost of a low yield of human insulin, when the process described in the European Patent Application with the Publication No. 0 474 213 is used. Additionally, it is evident that in this process separation can no longer be achieved if the concentration of acetylated insulin exceeds 5%. Furthermore, it is not possible using this process to obtain insulin that is virtually protease free.